上传用户：xrctutrubg资料价格：5财富值&&『』文档下载 ：『』&&『』学位专业：&关 键 词 ：&&&&&&权力声明：若本站收录的文献无意侵犯了您的著作版权，请点击。摘要:（摘要内容经过系统自动伪原创处理以避免复制，下载原文正常，内容请直接查看目录。）木质纤维素是最丰硕的可再生资本和可以或许转化成燃料的底物，将纤维质的生物量转化为可再生动力具有严重的意义。因为纤维素具有高度有序、慎密联合的晶体构造，又与半纤维素相联络，外周还由木质素包抄，要把它直接水解成可应用的葡萄糖相当艰苦。今朝对木质纤维素酶解多应用起源于丝状真菌的纤维素酶，但因为酶降解效力低下，酶解进程受产品克制，酶卵白不耐低温等，制约了其年夜范围运用。某些纤维素降解菌，如热纤梭菌具有高效的纤维素降解复合体一纤维小体，能有用降解纤维素，具有直接把生物资转化为乙醇和H2的才能，为应用一种微生物直接转化生物资的假想供给了能够。但在应用热纤梭菌停止乙醇发酵进程中常常伴随乙酸和丁酸等副产品的发生，并且终究产品的最高积聚浓度仅为3%阁下，只及酿酒酵母与发酵单胞菌属的25～30%。应用DNA重组技巧从新设计糖酵解门路，阻断副产品的构成道路，消除乙醇生物分解的代谢隔绝感化，同时进步细胞对高浓度乙醇的耐受性，是高产乙醇的梭菌属工程菌构建的重要内容。低温厌氧菌还具有产H2的才能。氢气具有热值高，无净化的特色。生物产氢包含光合产氢和发酵产氢。发酵产氢须要装备简略，不需光照，但H2的摩尔产量低和原资料价钱高还是未处理的成绩。发酵产氢只能应用无机物降解的15%能量。是以进步H2的产量将是往后研讨的重点。别的，也可应用两步法，将发酵产氢与光合产氢或与产甲烷联合，进步对生物资的应用效力。应用纤维素或废水产氢，可下降原料的本钱。本论文经由过程研讨嗜热厌氧纤维素降解菌的降解特征，对其纤维素酶系和调控机制有更深刻的懂得，同时可应用其可以或许转化纤维素生成氢气的特征，研讨纤维素发酵产氢的可行性。本论文的重要研讨内容以下1。嗜热厌氧纤维素降解菌的挑选与判定以纤维素为独一碳源，对嗜热厌氧纤维素降解菌停止富集。采取纤维素粉和纤维二糖滚管法对富集后的菌群进一步纯化，获得纤维素降解菌JN4和其伴生菌GD17。JN4能有用降解微晶纤维素和滤纸，也可应用玉米芯粉、玉米秸粉、木糖渣等自然底物作为碳源发展，GD17在富集液中数目较多，能应用纤维二糖、葡萄糖和木糖发展。经由过程形状不雅察，心理生化特征和16S rDNA剖析停止判定，肯定JN4为热纤梭菌（Clostridium thermocellum），GD17为热解糖低温厌氧杆菌（Thermoanaerobacterium thermosaccharolyticum）。2。C。thermocellum JN4和伴生菌T。thermosaccharolyticum GD17的发展和产氢特征以纤维二糖为碳源，肯定了C。thermocellum JN4和T。thermosaccharolyticumGD17的最适发展温度为55～60℃，并停止了发展曲线的测定。应用GC和HPLC对发酵产品的剖析注解，C。thermocellum JN4分化纤维素生成H2、乙酸、乙醇和乳酸，T。thermosaccharolyticum GD17可应用各类复原糖生成乙酸、乙醇、乳酸和丁酸。C。thermocellum JN4可降解微晶纤维素、滤纸，未经预处置的玉米秸粉和玉米芯粉。对C。thermocellum JN4的胞外卵白酶活测定注解，C。thermocellum JN4以微晶纤维素为碳源发展时，发酵液具有CMC酶活、磷酸收缩纤维素酶活和微晶纤维素酶活。以纤维二糖为碳源时，C。thermocellum JN4的H2产率(YH2)约为0。7mol H2/mol二糖；以纤维二糖、葡萄糖和木糖为碳源时，T。thermosaccharolyticum GD17的H2产率(YH2)分离为4mol H2/mol纤维二糖，2。2mol H2/mol葡萄糖和1。7mol H2/mol木糖。3。C。thermocellum JN4零丁造就和与T。thermosaccharolyticum GD17混杂造就应用纤维素发展和产氢的动力学研讨以微晶纤维素为碳源，研讨了厌氧管内C。thermocellum JN4零丁造就和与T。thermosaccharolyticum GD17混杂造就的发展和产氢动力学。成果注解零丁造就C。thermocellum JN4可有用降解0。5%微晶纤维素，发展前期有纤维二糖、葡萄糖的积聚，乳酸、乙酸和乙醇的量逐步增长，到60 h到达稳固，产H2才能为0。8 mol H2/mol葡萄糖。参加伴生菌T。thermosaccharolyticum GD17，与Cthermocellum JN4配合造就，可将C。thermocellum JN4发生的纤维二糖和葡萄糖完整应用，乙酸和乙醇产量与C。thermocellum JN4零丁接种比拟变更不年夜，有端岵?？并在60 h后仍有增加，乳酸发生在60 h以后开端降低，终究完整检测不到，而混杂菌的产H2才能进步到1。8 mol H2/mol葡萄糖。4。2L反响器中C。thermocellum JN4零丁造就和与T。thermosaccharolyticumGD17混杂造就应用纤维素的发展和产氢情形用克己的2L厌氧反响器在不掌握pH的情形下，以滤纸为碳源零丁造就C。thermocellum JN4，产氢量约为18mmol/1造就基，其它发酵产品有乙酸、乳酸和乙醇，个中乙醇的含量较高，到达24 mmol/1造就基以上，显示该菌有改革成为纤维素乙醇临盆菌的能够。与用厌氧管造就分歧，发酵液中没检测到纤维二糖和葡萄糖，注解扩展造就较有益于JN4的发展和代谢。C。thermocellum JN4与T。thermosaccharolyticum GD17混杂造就，在不掌握pH的情形下，产氢量约为35mmol/1造就基，其它发酵产品有乙酸、乳酸、丁酸和乙醇。乳酸含量在50h达到最高，然后逐步削减，同时丁酸含量逐步增长，与厌氧管造就成果类似。掌握pH在pH6。0一6。5，产氢量约为25mmol/1造就基，比不掌握pH要少。其它发酵产品中，乳酸含量最高，这注解pH在6。0一6。5的情形下，更有益于乳酸的生成，形成产氢量的削减。假如延伸造就时光，T。thermosaccharolyticum GD17可应用乳酸，会有更多的H2发生。5。C。thermocellum JN4外切葡聚糖酶基因celS的原核表达以C。thermocellum JN4的基因组为模板，以C。thermocellum ATCC 27405的celS基由于参考设计引物，PCR扩增获得的序列与Genbank的已知序列停止比对，成果注解扩增取得的片断为celS基因，且与Clostridium thermocellum ATCC27405的celS基因唯一3个碱基的差别。将C。thermocellum JN4的celS基因和质粒pET一22b衔接，构建表达载体，并应用年夜肠杆菌BL21（DE3）停止CelS的异源表达，获得的重组卵白rCelS具有水解磷酸收缩纤维素的才能。6。C。thermocellum JN4外切葡聚糖酶基因CelS的定点渐变联合卵白质构造生物学信息，对C。thermocellum JN4的rCelS卵白催化中间的相干位点停止D255N和Y351F定点渐变和原核表达，成果注解定点渐变后的两种rCelS卵白均没有水解磷酸收缩纤维素的才能。注解Asp255有能够作为狭义碱基团在外切葡聚糖酶CelS的催化反响中与狭义酸基团Glu87停止翻转型催化反响。Tyr351是CelS的守旧位点，将其酿成Phe能够对CelS的构造与催化功效形成影响。Abstract:Cellulose is the most abundant renewable resources and can be converted into a fuel substrate, the biomass into renewable energy has a serious significance. Because of the highly ordered, cheek by jowl with crystal structure and semi cellulose and phase contact with cellulose, peripheral also by lignin outflank to put it directly hydrolysis and the application of glucose is quite difficult. Current of cellulose solution of multi application origin in filamentous fungi, cellulase, but because of the low efficiency of degrading enzyme, enzymatic hydrolysis process by product restraint, albumen enzyme not resistant to low temperature, limits the scope of the eve of the use. Some cellulose degrading bacteria, such as Clostridium thermocellum has efficiency of cellulose degradation complex body of a fiber, effectively degrade cellulose, is directly to the biological information into ethanol and H2 can, for the application of a microbial direct conversion of raw materials of the imaginary provides the ability to. But in the application of Clostridium thermocellum stop ethanol fermentation process is often accompanied by the occurrence of acetic acid and butyric acid and other by-products, and eventually products of the highest accumulation concentration is only 3% of your, and wine yeast and fermentation Aeromonas sp. 25 ~ 30%. Application of recombinant DNA techniques from the design of a new sugar fermentation solution channels, blocking a byproduct of a road to eliminate biological decomposition of ethanol metabolism isolation effect, also improve cell tolerance to high concentration of ethanol is ethanol producing Clostridium is an important content of the construction of engineering bacteria. Low temperature anaerobic bacteria also have the ability to produce H2. Hydrogen has a high calorific value, non purification characteristics. Hydrogen production from bio hydrogen containing hydrogen production and hydrogen production. The production of hydrogen requires a simple equipment, no light, but the low molar yield of H2, and the price of raw materials is still not processed. Hydrogen production by fermentation can only be applied to the 15% energy of the degradation of inorganic compounds. Is to improve the production of H2 will be the focus of future research. In addition, two steps can be used to combine the fermentation of hydrogen production with hydrogen production or the production of methane. Use of cellulose or waste water to produce hydrogen, can reduce the cost of raw materials. This paper through research eosinophilic degradation characteristics of the thermophilic anaerobic cellulolytic bacteria, more profound understanding of the cellulases and regulation mechanism, and its application may change the characteristics of cellulose to produce hydrogen, the research of cellulose fermentation hydrogen production feasibility. The important research contents of this paper are as follows: 1. The selection and determination of thermophilic anaerobic cellulose decomposing bacteria were the only carbon source, and the concentration of thermophilic anaerobic cellulose decomposing bacteria was stopped. Cellulose powder and fiber two were taken to further purification of the bacteria after enrichment, and the cellulose degradation bacteria JN4 and its associated bacteria GD17 were obtained. Jn4 can useful degradation of microcrystalline cellulose and filter paper, can also be used corn cob powder and corn stalk powder, xylose residue and other natural substrate development as the carbon source and gd17 enrichment in the liquid in a large number of, application of fiber sugar, glucose and xylose development. Through shape observations, physiological and biochemical characteristics and 16S rDNA analysis judge, certainly jn4 Clostridium thermocellum and gd17 pyrolytic sugar low temperature anaerobic bacillus (Thermoanaerobacterium thermosaccharolyticum). 2. C. JN4 thermocellum and associated bacteria T. GD17 thermosaccharolyticum development and hydrogen production characteristics to the fiber two sugar as a carbon source, affirmed the C. JN4 thermocellum and T. The optimum development temperature of thermosaccharolyticumGD17 is 55 ~ 60 degrees C, and the development curve is stopped. Application of HPLC and GC on the analysis of the fermentation product notes, C. JN4 thermocellum to generate H2, acetic acid, ethanol and lactic acid, T. GD17 thermosaccharolyticum can be used to produce various types of recovered sugar, acetic acid, ethanol, lactic acid and butyrate. C. Thermocellum JN4 can degrade microcrystalline cellulose, filter paper, without pretreatment of corn stalk powder and corn cob powder. On C. Determination of extracellular protease thermocellum annotations live JN4, C. JN4 thermocellum in the development of microcrystalline cellulose as the carbon source, the fermentation broth with CMC enzyme activity, phosphoric acid contraction of cellulase activity and microcrystalline cellulose activity. When the fiber two sugar as carbon source, C. JN4 H2 thermocellum yield (YH2) is about 0. H2/mol 7 fiber two sugar, glucose and xylose as carbon source, T. GD17 H2 thermosaccharolyticum yield (YH2) was isolated from H2/mol 4mol fiber two sugar, 2. H2/mol 2mol glucose and 1. H2/mol 7mol xylose. 3. C. JN4 thermocellum zero Ding made and with T. GD17 thermosaccharolyticum hybrid to create applications for the development of cellulose and hydrogen production kinetics of microcrystalline cellulose as carbon source, the study of the anaerobic tube C. JN4 thermocellum zero Ding made and with T. Development and hydrogen production kinetics of GD17 thermosaccharolyticum hybrid. Results notes zero Ding made on C. JN4 thermocellum can be useful for degradation of 0. 5% microcrystalline cellulose, the development of fiber two sugar, glucose accumulation, the amount of lactic acid, acetic acid and ethanol has gradually increased, reaching a stable 60 h, the yield of H2 can be 0. 8 H2/mol mol glucose. Participate in associated bacteria T. Thermosacc目录:目录4-9摘要9-13ABSTRACT13-16符号说明及缩略词17-18第一章 绪论18-42&&&&1.1 纤维素酶概述18-20&&&&1.2 纤维小体的研究进展20-29&&&&&&&&1.2.1 C.thermocellum纤维小体酶组分组成和结构21-22&&&&&&&&1.2.2 C.thermocellum纤维小体纤维素酶活性的特点22&&&&&&&&1.2.3 C.thermocellum外切葡聚糖酶CelS22-23&&&&&&&&1.2.4 C.thermocellum纤维素酶、半纤维素酶合成的调节机制23-26&&&&&&&&1.2.5 人工纤维小体26-29&&&&1.3 嗜热厌氧纤维素降解细菌工业化潜力的研究29-39&&&&&&&&1.3.1 C.thermocellum产乙醇的研究进展29-31&&&&&&&&1.3.2 梭菌联合培养31-33&&&&&&&&1.3.3 利用DNA重组技术来解决选择性难题33-34&&&&&&&&1.3.4 梭菌发酵产氢的研究34-39&&&&1.4 本研究的目的与主要工作内容39-42第二章 嗜热厌氧纤维素降解菌的筛选与鉴定42-58&&&&引言42&&&&2.1 材料和方法42-46&&&&&&&&2.1.1 样品来源42-43&&&&&&&&2.1.2 培养基43&&&&&&&&2.1.3 主要仪器与试剂43&&&&&&&&2.1.4 厌氧培养方法43-44&&&&&&&&2.1.5 菌种的富集和分离纯化44&&&&&&&&2.1.6 菌种鉴定44-46&&&&2.2 结果与分析46-55&&&&&&&&2.2.1 厌氧培养方法的建立46&&&&&&&&2.2.2 菌种的富集46-47&&&&&&&&2.2.3 菌群的16S rDNA序列分析47-50&&&&&&&&2.2.4 菌种的培养特征与个体形态50-55&&&&2.3 讨论55-57&&&&小结57-58第三章 嗜热厌氧纤维素降解菌C.thermocellum JN4和伴生菌T.thermosaccharolyticum GD17的生长及产氢特性58-78&&&&引言58-59&&&&3.1 材料和方法59-62&&&&&&&&3.1.1 菌株59&&&&&&&&3.1.2 培养基59&&&&&&&&3.1.3 主要仪器与试剂59&&&&&&&&3.1.4 厌氧培养方法59&&&&&&&&3.1.5 生长曲线的测定59-60&&&&&&&&3.1.6 最适生长温度的确定60&&&&&&&&3.1.7 pH的测定60&&&&&&&&3.1.8 发酵产物的分析60&&&&&&&&3.1.9 氢气产率(Y_(H2))的计算60&&&&&&&&3.1.10 蛋白质电泳和活性染色60-61&&&&&&&&3.1.11 胞外蛋白酶活测定61-62&&&&3.2 结果与分析62-74&&&&&&&&3.2.1 最适生长温度的确定63&&&&&&&&3.2.2 菌体生长测定63-64&&&&&&&&3.2.3 纤维素降解菌C.thermocellum JN4和伴生菌T.thermosaccharolyticum GD17的产氢特性64-67&&&&&&&&3.2.4 C.thermocellum JN4和T.thermosaccharolyticum GD17以不同浓度纤维素底物为碳源时的生长67-68&&&&&&&&3.2.5 C.thermocellum JN4和T.thermosaccharolyticum GD17对木聚糖的降解68-69&&&&&&&&3.2.6 C.thermocellum JN4和T.thermosaccharolyticum GD17对天然纤维素类物质的利用69-71&&&&&&&&3.2.7 C.thermocellum JN4的胞外蛋白及其纤维素酶活分析71-74&&&&3.3 讨论74-75&&&&小结75-78第四章 C.thermocellum JN4和伴生菌T.thermosaccharolyticum GD17利用纤维素生长和产氢的动力学研究78-96&&&&引言78-79&&&&4.1 材料和方法79-81&&&&&&&&4.1.1 菌株79&&&&&&&&4.1.2 培养基79&&&&&&&&4.1.3 主要仪器与试剂79&&&&&&&&4.1.4 厌氧培养方法79&&&&&&&&4.1.5 蛋白浓度的测定79&&&&&&&&4.1.6 碳源利用79-80&&&&&&&&4.1.7 pH的测定80&&&&&&&&4.1.8 发酵产物的分析80&&&&&&&&4.1.9 氢气产率(Y_(H2))的计算80&&&&&&&&4.1.10 2L厌氧反应器的设计与使用80-81&&&&4.2 结果与分析81-91&&&&&&&&4.2.1 菌体的生长与碳源的利用81-82&&&&&&&&4.2.2 pH的变化82-84&&&&&&&&4.2.3 H_2的产生84-85&&&&&&&&4.2.4 还原糖、有机酸和乙醇的生成85-88&&&&&&&&4.2.5 2L反应器的设计与使用88-91&&&&4.3 讨论91-94&&&&小结94-96第五章 嗜热厌氧纤维素降解菌C.thermocellum JN4外切葡聚糖酶基因CelS的原核表达和定点突变96-116&&&&引言96-97&&&&5.1 材料和方法97-103&&&&&&&&5.1.1 菌株和质粒97&&&&&&&&5.1.2 培养基97&&&&&&&&5.1.3 试剂、工具酶与仪器97-98&&&&&&&&5.1.4 C.thermocellum JN4外切葡聚糖酶基因celS的克隆及序列分析98-100&&&&&&&&5.1.5 表达载体的构建100&&&&&&&&5.1.6 重组蛋白的诱导表达与复性100-102&&&&&&&&5.1.7 酶活测定方法102&&&&&&&&5.1.8 CelS基因的D255N和Y351F点突变102-103&&&&5.2 结果与分析103-111&&&&&&&&5.2.1 基因组DNA的提取103&&&&&&&&5.2.2 C.thermocellum JN4 celS基因PCR扩增103&&&&&&&&5.2.3 测序结果和已知序列差异103-106&&&&&&&&5.2.4 表达载体的构建106-107&&&&&&&&5.2.5 重组蛋白的诱导表达与复性107-108&&&&&&&&5.2.6 rCelS蛋白的酶活性测定108-110&&&&&&&&5.2.7 celS基因的D255N和Y351F点突变110-111&&&&5.3 讨论111-113&&&&小结113-116全文总结与展望116-118参考文献118-138攻读学位期间发表的学术论文138-140致谢140-141学位论文评阅及答辩情况表141-142附件142-156分享到：相关文献|热纤维素梭菌内切葡聚糖苷酶基因在枯草芽孢杆菌的表达_图文_百度文库
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